Field of the Invention
The present invention relates generally to immunoreagents, and more specifically to Rift Valley fever Virus (RVFV) glycoproteins and their use as vaccine components and as moieties for disease diagnosis and detection, including methods of using such glycoproteins.
Background Information
Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen that causes high morbidity and mortality in both livestock and humans. The virus has caused outbreaks in ruminants and humans in Africa and the Arabian Peninsula and is classified as a select agent and risk group-3 pathogen by the Centers for Disease Control and Prevention (CDC) and the United States department of Agriculture (USDA).
In ruminant livestock, Rift Valley fever (RVF) is characterized by high mortality in young animals, notably in lambs, fetal malformations and widespread abortion storms; sheep are the most susceptible, with neonatal mortalities approaching 100%. Human infections are often characterized by benign fever but in small proportion of individuals could lead to more serious complications such as retinitis, encephalitis, neurological disorders, hepatic necrosis, or fatal hemorrhagic fever. Although human fatal hemorrhagic cases have been historically estimated at 2% in infected individuals, case fatality rates have increased significantly in recent years as high as 20% including the recent outbreak in Mauritania.
There is increasing demand for sensitive and safe diagnostic tests and efficacious vaccines for zoonotic pathogens, including RVFV, to protect human and animal health. The recent spread of RVFV beyond its traditional endemic boundaries in Africa to the Arabian Peninsula (Jupp and Cornet 1988, Abdo-Salem, et al. 2011, Ikegami and Makino 2009) has resulted in increased interest for RVFV vaccines, rapid diagnostics and associated immunoreagents.
RVFV belongs to the genus Phlebovirus within the family Bunyaviridae, which includes over 350 named isolates. It has a tripartite single-stranded RNA genome of negative polarity consisting of small (S), medium (M) and large (L) RNA segments. The M segment encodes for the two structural glycoproteins, Gn/Gc, the 78-kDa protein and the non-structural protein, NSm; and the S segment for the nucleocapsid protein (N) protein and the non-structural protein, NSs. The L-segment encodes for the RNA-dependent RNA polymerase. The N and L proteins are required for viral RNA synthesis; and the NSs protein is the major virulence factor and has been shown to inhibit host transcriptional immune response through generalized transcription downregulation including repression of IFN-β and degradation of protein kinase R. The NSm protein functions to suppress virus-induced apoptosis. The glycoproteins, Gn and Gc, are surface proteins that play a role in virus attachment to initiate infection and have been shown to carry epitopes that elicit the production of neutralizing antibodies, a correlate of protective immunity.
The nucleocapsid (N) protein is the most abundant and highly immunogenic component of the RVF virion and has been used for development of diagnostic assays for detection of RVFV specific antibodies in human and animal sera. Although the N protein is shown to be highly conserved among members of the Bunyaviridae family, a previous indirect ELISA based on the recombinant protein did not show cross-reactivity with other African phleboviruses that could hamper the reliability of using this protein in assays for serodiagnosis of RVFV infection. However, the N protein did show serological cross-reactivity with an unidentified agent among some sear from US and Canadian sheep.
There are currently no RVFV vaccines fully approved for use outside its endemic area in Africa and the Arabian Peninsula. Given the potential for viral spread elsewhere including the mainland US, there is an urgent need for a safe and efficacious vaccine. Attributes essential for a vaccine for use in non-endemic areas include safety and the ability to generate a rapid (with primary vaccination) protective immune response in a susceptible host. At present, RVFV in endemic regions is controlled in livestock using live-attenuated Smithburn strain or inactivated whole virus. The Smithburn vaccine is highly immunogenic but is teratogenic in pregnant sheep and cattle. The whole-virus formalin inactivated vaccines are safe but less immunogenic. Other live-vaccine candidates under evaluation are Clone 13 (licensed for use in South Africa), a natural attenuated isolate from a benign RVF case in the Central African Republic, and MP12, a chemically attenuated virus derived from ZH548, an Egyptian wild-type isolate. The immunogenicity and pathogenicity of these candidate vaccines have been evaluated in various animal species; and although both vaccine candidates showed promising results, the MP12 induced fetal malformations during the first trimester. However, a recent study reported the absence of fetal malformations in pregnant ewes inoculated with the virus. Strategies to develop RVFV vaccines include subunit, DNA, virus-like particles, virus replicon particles, virus-vectored, modified live vaccines using reverse genetic engineering, live attenuated, and inactivated whole virus vaccines.
Although some of these vaccines have shown promising results, their immunogenicity and efficacy have either not been determined in a natural host species or have not been shown to induce protective neutralizing antibody titer in single immunization. On the other hand, production of live-vaccines requires high level of biosafety; and their use is associated with potential side effects. Therefore, the general availability of a safe, inexpensive vaccine with DIVA compatibility will be extremely valuable to non-endemic countries outside Africa including the US.
At the present, diagnosis of RVFV infection is achieved using various techniques including virus isolation, antigen detection, nucleic acid amplification techniques, and detection of RVFV specific antibodies. The use of virus isolation is not user-friendly, takes an extended period of time and is also unsafe for laboratory personnel; antigen or nucleic acid detection in the blood of animals only works in cases of host viremia, which in the case of RVFV infection, is a narrow viremic window, lasting on average about 3 days.
The classical methods for detection of antibodies to RVFV include various forms of virus neutralization and haemagglutination inhibition tests. Disadvantages of these techniques include health risk to laboratory personnel, as well as restrictions to high biocontainment laboratories for their use outside RVF endemic areas. On the other hand, application of ELISA to detect IgG antibody to RVFV relied largely on the use of inactivated whole virus lysate, which is also associated with potential health risks.
What is needed are potent virus neutralizing antibody response inducers as efficacious vaccines against RVFV, including immunoreagents that may serve as moieties for effective RVFV detection and disease diagnosis.